Abstract
In the budding yeast Saccharomyces cerevisiae the protein phosphatase Sit4 and four associated proteins (Sap4, Sap155, Sap185, and Sap190) mediate G1 to S cell cycle progression and a number of signaling events controlled by the target of rapamycin TOR signaling cascade. Sit4 and the Sap proteins are ubiquitously conserved and their human orthologs, PP6 and three PP6R proteins, share significant sequence identity with their yeast counterparts. However, relatively little is known about the functions of the PP6 and PP6R proteins in mammalian cells. Here we demonstrate that the human PP6R proteins physically interact with Sit4 when expressed in yeast cells. Remarkably, expression of PP6R2 and PP6R3 but not expression of PP6R1 rescues the growth defect and rapamycin hypersensitivity of yeast cells lacking all four Saps, and these effects require Sit4. Moreover, PP6R2 and PP6R3 enhance cyclin G1 gene expression and DNA synthesis, and partially abrogate the G1 cell cycle delay and the budding defect of the yeast quadruple sap mutant strain. In contrast, the human PP6R proteins only modestly support nitrogen catabolite gene expression and are unable to restore normal levels of eIF2α phosphorylation in the quadruple sap mutant strain. These results illustrate that the human PP6-associated proteins are capable of providing distinct rapamycin-sensitive and Sit4-dependent Sap functions in the heterologous context of the yeast cell. We hypothesize that the human Saps may play analogous roles in mTORC1-PP6 signaling events in metazoans.
Highlights
Protein dephosphorylation is an essential enzymatic activity through which cell signaling in response to diverse cues is processed and cellular events evoked by protein phosphorylation are mechanistically reversed
The human PP6R proteins are stably expressed in yeast cells and physically interact with Sit4
The striking conservation between the yeast Saps and the PP6R proteins prompted us to explore the functions of the PP6R proteins in yeast cells
Summary
Protein dephosphorylation is an essential enzymatic activity through which cell signaling in response to diverse cues is processed and cellular events evoked by protein phosphorylation are mechanistically reversed. In the yeast Saccharomyces cerevisiae the type 2A-like protein phosphatase catalytic subunit Sit functions downstream of the rapamycin-sensitive TOR complex 1 (TORC1) to govern responses to nutrients and events required for normal G1 to S phase cell cycle transition and budding [1,2]. In response to nutrient signals TORC1 modulates interaction of Sit with its essential effector Tap and thereby regulates translation, expression of the nitrogen catabolite-repressed (NCR) and retrograde response genes, and the Pkc cell integrity pathway [3,4,5,6]. Homologs of Sit and Tap, known as PP6 and alpha, respectively have been identified in mammalian cells. Rapamycin-sensitive functions have been ascribed to the PP2A-alpha protein phosphatase, a role for the PP6-alpha holoenzyme in mTORC1signaling has not yet been demonstrated [8,9,10]. PP6 shares 61% amino acid sequence identity with Sit and functionally complements sit mutations in S. cerevisiae and of the Sit homolog ppe in Schizosaccharomyces pombe [11]
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